It is estimated that between 20-25% of American adults (about 47 million) have metabolic syndrome, a complex condition associated with an increased risk of vascular disease. Metabolic syndrome is also known as Syndrome X, metabolic syndrome X, insulin resistance syndrome, or Reaven's syndrome. Metabolic syndrome is generally believed to be a combination of disorders that affect a large number of people in a clustered fashion. The symptoms and features of the syndrome include at least three of the following conditions: diabetes mellitus type II; impaired glucose tolerance or insulin resistance; high blood pressure; central obesity and difficulty losing weight; high cholesterol; combined hyperlipidemia; including elevated LDL; decreased HDL; elevated triglycerides; and fatty liver (especially in concurrent obesity). Insulin resistance is typical of metabolic syndrome and leads to several of its features, including glucose intolerance, dyslipidemia, and hypertension. Obesity is commonly associated with the syndrome as is increased abdominal girth, highlighting the fact that abnormal lipid metabolism likely contributes to the underlying pathophysiology of metabolic syndrome.
Metabolic syndrome was codified in the United States with the publication of the National Cholesterol Education Program Adult Treatment Panel III (ATP III) guidelines in 2001. On a physiologic basis, insulin resistance appears to be responsible for the syndrome. However, insulin resistance can be defined in a myriad of different ways, including impaired glucose metabolism (reduced clearance of glucose and/or the failure to suppress glucose production), the inability to suppress lipolysis in tissues, defective protein synthesis, altered cell differentiation, aberrant nitric oxide synthesis affecting regional blood flow, as well as abnormal cell cycle control and proliferation, all of which have been implicated in the cardiovascular disease associated with metabolic syndrome. At least at present, there is no obvious molecular mechanism causing the syndrome, probably because the condition represents a failure of one or more of the many compensatory mechanisms that are activated in response to energy excess and the accumulation of fat.
According to ATP III, the diagnosis of metabolic syndrome requires the presence of three or more of the following: elevated fasting triglycerides (greater than or equal to 150 mg/dl), low HDL cholesterol (less than 50 mg/dl in women, less than 40 mg/dl in men), hypertension (blood pressure greater than or equal to 130/85 mm Hg), increased waist circumference (due to excess visceral adiposity, greater than 35 inches in women, greater than 40 inches in men) and elevated fasting glucose (greater than or equal to 100 mg/dl). The presence of three components is not a perfect predictor of insulin resistance, and the World Health Organization has established somewhat different criteria that include microalbuminuria (i.e., slightly elevated albumin excretion in the urine), and some groups modify the ATP III criteria to include a body mass index (BMI) of greater than or equal to 30 kg/M2 and abnormal nonfasting glucose and lipid values. Regardless of the definition, the syndrome identifies a group of individuals at increased risk for vascular disease. In an analysis of the Third National Health and Nutrition Examination Survey (NHANES III) participants over the age of 50 with metabolic syndrome showed a coronary heart disease prevalence exceeding that of diabetes. NHANES II data indicate total mortality as well as death from coronary heart disease and cardiovascular disease are increased in adults with metabolic syndrome.
Individuals at risk for metabolic syndrome include those who exhibit central obesity with increased abdominal girth (due to excess visceral adiposity) of about more than 35 inches in women and more than 40 inches in men. Individuals at risk for metabolic syndrome also include those that have a BMI greater than or equal to 30 kg/m2 and may also have abnormal levels of nonfasting glucose, lipids, and blood pressure.
Obesity has become widespread with increases in prevalence across all developed nations (Bouchard, C (2000) N Engl J Med. 343, 1888-9). According to the Center for Disease Control (CDC), over 60% of the United States population is overweight, and greater than 30% are obese. For affected persons, the problem often begins in childhood, and continues for life. Major contributors are believed to be increased consumption of high calorie foods and a more sedentary life style. However, neither of these alone or together are sufficient to explain the rise in obesity and subsequent or concomitant obesity-related disorders, such as, e.g., type II diabetes mellitus, metabolic syndrome, hypertension, cardiac pathology, and non-alcoholic fatty liver disease. According to the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) approximately 280,000 deaths annually are directly related to obesity. The NIDDK further estimated that the direct cost of healthcare in the U.S. associated with obesity is $51 billion. In addition, Americans spend $33 billion per year on weight loss products. The prevalence of obesity continues to rise at alarming rates.
Gastrointestinal reflux disease (GERD) and related disorders including Barrett's esophagus (BE), adenocarcinoma of the esophagus (EAC), and adenocarcinoma of the gastric cardia, (which collectively can be termed gastro-esophageal junction adenocarcinomas (GEJAC), are also becoming increasingly important medical problems in the United States and other developed countries. For affected persons, the problem often begins in childhood, but more typically presents clinically in adulthood, and continues for life. These disorders have become widespread with increases in incidence and prevalence across all developed nations. The rise in the problem has been so rapid that the major effect must be environmental, and not genetic. Major contributors are believed to be exposure to excess gastric acidity and particular foods and medications. However, none of these can fully explain the rise in GERD and its related conditions across widespread population groups. An alternative explanation is that the GERD is due to the growing epidemic of obesity, but since many non-obese persons suffer from GERD, this also is insufficient to explain the explosive rise in GERD and related esophageal diseases.
Similarly, childhood-onset asthma and related disorders including allergic rhinitis (“hay fever”) and eczema (atopic dermatitis) are also becoming increasingly important medical problems in the United States and other developed countries. Asthma and related disorders have become widespread with increases in prevalence across all developed nations. Major contributors are believed to be exposure to environmental irritants, such as air pollution, tobacco smoke, and allergens, such as insect populations, and environmental microbes. However, none of these are sufficient to explain the rise in asthma and its related conditions across widespread population groups. An alternative explanation is that the lack of exposures to environmental microbes, such as those found in soil, in pets, and in farm animals is responsible for the rise in asthma (often called the “hygiene hypothesis”), but this too is insufficient to explain the explosive rise in asthma, especially that which begins in early childhood.
Although certain bacterial associations have been examined for these conditions, the role of bacterial microbiota in ailments such as asthma, obesity, GERD and certain related cancers, all of which have been on the rise in the 21st century, has not been clearly understood or appreciated. Thus, there remains a need for methods for diagnosing, treating and preventing conditions such as asthma, obesity, metabolic syndrome, GERD, gastro-esophageal junction adenocarcinomas (GEJAC), and related disorders.
The average human body, consisting of about 1013 cells, has about ten times that number of microorganisms. The ˜1014 microbes that live in and on each of our bodies belong to all three domains of life on earth—bacteria, archaea and eukarya. The major sites for our indigenous microbiota are the gastrointestinal tract, skin and mucosal surfaces such as nasal mucosa and vagina as well as the oropharynx. By far, the largest bacterial populations are in the colon. Bacteria make up most of the flora in the colon and 60% of the dry mass of feces. Probably more than 1000 different species live in the gut. However, it is probable that >90% of the bacteria come from less than 50 species. Fungi and protozoa also make up a part of the gut flora, but little is known about their activities. The skin also has a diverse microbiome, also with likely >1000 species, yet with major populations within a small number of species (Gao et al., Proc. Natl. Acad. Sci. USA 2007, 104(8):2927-2932). While the microbiota is highly extensive, it is barely characterized. Consequently, the Roadmap of the National Institutes of Health (NIH) includes the “Human Microbiome Project” to better characterize our microbial communities and the genes that they harbor (our microbiome) and better understand its relation to both human health and disease. Reviewed in Dethlefsen et al., Nature, 2007, 449:811-818; Turnbaugh et al., Nature, 2007, 449:804-810; Ley et al., Cell, 2006, 124:837-848.
Studies show that the relationship between gut flora and humans is not merely commensal (a non-harmful coexistence), but rather often is a mutualistic, symbiotic relationship. Although animals can survive with no gut flora, the microorganisms perform a host of useful functions, such as training the immune system, preventing growth of harmful species, regulating the development of the gut, fermenting unused energy substrates, metabolism of glycans and amino acids, synthesis of vitamins (such as biotin and vitamin K) and isoprenoids, biotransformation of xenobiotics, and producing hormones to direct the host to store fats. See, e.g., Gill et al., Science. 2006, 312:1355-1359; Zaneveld et al., Curr. Opin. Chem. Biol., 2008, 12(1):109-114; Guarner, Digestion, 2006, 73:5-12; Li et al., Proc. Natl. Acad. Sci. USA, 2008, 105:2117-2122; Hooper, Trends Microbiol., 2004, 12:129-134; Mazmanian et al., Cell, 2005, 122:107-118; Rakoff-Nahoum et al., Cell, 2004, 118:229-241. It is therefore believed that changes in the composition of the gut microbiota could have important health effects (Dethlefsen et al., PLoS Biology, 2008, 6(11):2383-2400). Indeed, a correlation between obesity and changes in gut microbiota has been observed (Ley et al., Proc Natl Acad Sci USA, 2005; 102:11070-11075; Bäckhed et al., Proc Natl Acad Sci USA, 2004; 101:15718-15723). Furthermore, in certain conditions, some microbial species are thought to be capable of directly causing disease by causing infection or increasing cancer risk for the host (O'Keefe et al., J. Nutr. 2007; 137:175S-182S; McGarr et al., J Clin Gastroenterol., 2005; 39:98-109).
Substantial number of species in vertebrate microbiota is very hard to culture and analyze via traditional cultivation-based studies (Turnbaugh et al., Nature, 2007, 449:804-810; Eckburg et al., Science, 2005, 308:1635-1638). In contrast, broad-range PCR primers targeted to highly conserved regions makes possible the amplification of small subunit rRNA gene (16S rDNA) sequences from all bacterial species (Zoetendal et al., (2006) Mol Microbial 59, 1639-1650), and the extensive and rapidly growing 16S rDNA database facilitates identification of sequences to the species or genus level (Schloss and Handelsman, (2004) Microbiol Mol Biol Rev 68, 686-691). Such techniques can also be used for identifying bacterial species in complex environmental niches (Smit et al., (2001) Appl Environ Microbiol 67, 2284-2291), including the human mouth, esophagus, stomach, intestine, feces, skin, and vagina, and for clinical diagnosis (Harris and Hartley, (2003) J Med Microbiol 52, 685-691; Saglani et al., (2005) Arch Dis Child 90, 70-73).
Much of the microbiota is conserved from human to human, at least at the level of phylum and genus (for a general description of human microbiota see, e.g., Turnbaugh et al., Nature 2007; 449:804-810; Ley et al., Nature 2006; 444:1022-1023; Gao et al., Proc Natl Acad Sci USA 2007; 104:2927-32; Pei et al., Proc Natl Acad Sci USA 2004; 101:4250-4255; Eckburg et al., Science 2005; 308:1635-1638; Bik et al., Proc Natl Acad Sci USA 2006; 103:732-737). A major source of the human microbiota is from one's mother (for a summary of typical maternal colonization patterns see, e.g., Palmer et al., Plos Biology 2007; 5:e177; Raymond et al., Emerg Infect Dis 2004; 10:1816-21), and to a lesser extent from one's father and siblings (for examples of typical colonization patterns see, e.g., Raymond et al., Emerg Infect Dis 2004; 10:1816-21; Raymond et al., Plos One 2008; 3:e2259; Goodman et al., Am J Epidemiol 1996; 144:290-299; Goodman et al., Lancet 2000; 355:358-362). However, many of the natural mechanisms for the transmission of these indigenous organisms across generations and between family members have diminished with socioeconomic development. The impediments include: childbirth by caesarian section, reduced breast-feeding, smaller family size (fewer siblings), reduced household crowding with shared beds, utensils, in-door plumbing.
Effective antibiotics were discovered in the early-mid 20th century and came into wide use after World War II. Antibiotic use has increased dramatically with rates approximating one course of antibiotics per year in the average child in the USA (for a summary of US antibiotic courses in a year, see, e.g., McCaig et al., JAMA 2002; 287:3096-3102).
Antibiotic use places selective pressure on the microbiota, in particular selecting for the long-term persistence of resistant organisms (such persistence is described in Levy, Sci Am 1998; 278:46-53). Antibiotic resistance may be intrinsic or secondary to acquired genetic elements, but marker organisms (and genes) may be used to observe the phenomenon (examples of such markers may be found in, e.g., Sjölund et al., Annals of Internal Medicine 2003; 139: 483-487; Sjölund et al., Emerging Infectious Diseases 2005:11:1389-1393).
Increased exposure to antibiotics in the first year of life has been associated with increased risk of developing asthma by seven years of age (Kozyrskyj et al., Chest. 2007; 131:1753-9). The effects are not specific to a single class of antibiotics, but involve many different agents. Additionally, the risk of asthma and related disorders has previously been inversely associated with the risk of having gastric colonization by H. pylori, as ascertained from serological tests (see, e.g., Reibman et al., Presented at ATS 2005; Chen and Blaser, Arch Intern Med 2007; 167:821-827; Chen and Blaser, J. Infect. Dis. 2008; 198:553-60; Blaser et al., Gut 2008; 57:561-7). The risk appears primarily limited to childhood onset asthma and related conditions.
The acute effects of antibiotic treatment on the native gut microbiota range from self-limiting “functional” diarrhea to life-threatening pseudomembranous colitis (Beaugerie and Petit, Best Pract Res Clin Gastroenterol. 2004; 18:337-352; Wilcox, Best Pract Res Clin Gastroenterol. 2003; 17:475-493). The long-term consequences of such perturbations for the human-microbial symbiosis are more difficult to discern, but chronic conditions such as asthma and atopic disease have been associated with childhood antibiotic use and an altered intestinal microbiota (see, e.g., Marra et al.; Chest. 2006; 129:610-618; Noverr and Huffnagle, Clin Exp Allergy. 2005; 35:1511-1520; Prioult and Nagler-Anderson; Immunol Rev. 2005; 206:204-218).
It has been known for more than 50 years that the administration of low doses of antibiotics promotes the growth of farm animals. As a result, the largest use of antibiotics and other antimicrobial substances is on the farm, where they are fed in low doses to large numbers of animals used for food production. Additionally, the following observations regarding antibiotic use are appreciated:                1. feeding low (subtherapeutic) doses of antimicrobials promotes weight gain (often 5-10% of total weight) of animals used for food production (See, e.g., Jukes, Bioscience 1972; 22: 526-534; Jukes (1955) Antibiotics in Nutrition. New York, N.Y., USA: Medical Encyclopedia; Feighner and Dashkevicz, Appl. Environ. Microbiol., 1987, 53: 331-336; McEwen and Fedorka-Cray, Clin. Infect. Dis., 2002, 34 (Suppl 3): S93-S106);        2. the effects are broad across vertebrate species, involving at least mammals (cattle, swine, sheep), and birds (chickens and turkeys);        3. the effects can be realized by oral administrations of the agents, suggesting that the microbiota of the gastrointestinal tract is a major target;        4. the effects are due to many different classes of antimicrobial agents (including macrolides, tetracyclines, penicillins);        5. anti-fungal agents do not produce the effect;        6. the effects can be observed at many different stages in the growth and development of young animals.        
The mechanism for this widespread phenomenon has not been established but because of the activity of anti-bacterial but not anti-fungal agents, it can be ascertained to be anti-bacterial.
The vertebrate gastrointestinal tract has a rich component of cells involved in immune responses. The nature of the microbiota colonizing experimental animals or humans affects the immune responses of the populations of reactive host cells (see, e.g., Ando et al., Infection and Immunity 1998; 66:4742-4747; Goll et al., Helicobacter. 2007; 12:185-92; Lundgren et al., Infect Immun. 2005; 73:523-531).
The vertebrate gastrointestinal tract also is a locus in which hormones are produced. In mammals, many of these hormones related to energy homeostasis (including insulin, glucagon, leptin, and ghrelin) are produced by organs of the gastrointestinal tract (see, e.g., Mix et al., Gut 2000; 47:481-6; Kojima et al., Nature 1999; 402:656-60; Shak et al., Obesity Surgery 2008; 18(9):1089-96; Roper et al., Journal of Clinical Endocrinology & Metabolism 2008; 93:2350-7; Francois et al., Gut 2008; 57:16-24; Cummings and Overduin, J Clin Invest 2007; 117:13-23; Bado et al., Nature 1998; 394:790-793).
Changing of the microbiota of the gastrointestinal tract appears to affect the levels of some of these hormones (see, e.g., Breidert et al., Scand J Gastroenterol 1999; 34:954-61; Liew et al., Obes. Surg. 2006; 16:612-9; Nwokolo et al., Gut. 2003; 52, 637-640; Kinkhabwala et al., Gastroenterology 132:A208). The hormones affect immune responses (see, e.g., Matarese et al., J Immunol 2005; 174:3137-3142; Matsuda et al., J. Allergy Clin. Immunol. 2007; 119, S174) and adiposity (see, e.g., Tschop et al., Nature 2000; 407:908-13).